Complete differentiation between enkephalinase and angiotensin-converting enzyme inhibition by retro-thiorphan.

Abstract

Thiorphan, N-[(R,S)-3-mercapto-2-benzylpropanoyliglycine is a highly potent inhibitor (K; = 3.5 nM) of "enkephalinase," a metalloendopeptidase cleaving the Gly-Phe bond (positions 3 and 4) of enkephalins in brain tissue. In accordance with this property, thiorphan displays antinociceptive activity after systemic administration. However, thiorphan also inhibits to a lesser extent (K; = 140 nM) the widely distributed angiotensin-converting enzyme, a carboxydipeptidase implicated in blood pressure regulation. Therefore, in view of an eventual clinical use of enkephalinase inhibitors, it was very important to develop fully specific compounds. Such derivatives were obtained taking into account that N-methylation of the ultimate amide bond of dipeptides strongly decreases enkephalinase affinity without affecting angiotension-converting enzyme recognition, whereas retro-inversion of the amide bond leads to the inverse effect. Thus, the retro-inverso dipeptide (R)-H2N-CH(CH24)-NHCO-CH2-CO2H exhibits an inhibitory potency on enkephalinase (IC50 12 jM) close to that of the natural dipeptide L-Phe-Gly (IC50 3 jIM). This result shows the topological analogy between the crucial components involved in enkephalinase recognition both in active dipeptides and structurally related retro-inverso isomers. Taking into account these observations, retro-thiorphan, (R,S)-HS-CH2-CH-(CH20)-NHCO-CH2-COOH, was prepared. As compared to thiorphan, the retro isomer is 50% as potent (Ki = 6 nM) on enkephalinase but displays a drastic loss of potency on angiotension-converting enzyme (IC50 > 10,000 nM). This specificity was interpreted as a consequence of differences in the stereochemical constraints involving enzyme-inhibitor hydrogen bonding. This hypothesis is supported by reported crystallographic studies on related enzymes such as thermolysin and carboxypeptidase A. As expected, retro-thiorphan exhibits about the same analgesic potency as thiorphan on the hot plate and writhing tests in mice. Therefore, the topological concept of retro-inverso isomers could be extended to other enkephalinase inhibitors, allowing the design of potent and highly selective compounds occurring as new classes of analgesic and psychoactive agents.An important approach in the search for antinociceptive agents is to prevent degradation of the endogenous morphine-like peptides, enkephalins (1, 2), at the synaptic level. The characterization of a membrane.bound metalloendopeptidase called enkephalinase (3)(4)(5), which seems to be specifically involved in cleavage of the Gly-Phe (positions 3 and 4) bond of [Met]enkephalin (Tyr-Gly-Gly-Phe-Met) or [Leu]enkephalin (Tyr-GlyGly-Phe-Leu), made this approach possible. A highly potent enkephalinase inhibitor, thiorphan, N-[(R,S)-3-mercapto-2-benzylpropanoyl]glycine [Ki = 3.5 nM (6, 7)], was rationally designed taking into account the occurrence in this peptidase of (i) a Zn atom in its catalytic site able to coordinate a thiol group with a strong affinity (7, 8); (ii) a Sj' subsite exhibiting a specificity for aro...

The partial retro–inverso modification: A road traveled together

“…Guided by the active site model and following the topological concept underlying the PMRI transformation, this laboratory designed an end-group modified RI dipeptide (R)-H-gPhemGly-OH that inhibited enkephalinase with potency similar to the natural dipeptide L-H-Phe-Gly-OH (IC 50 Ϸ12 vs. 3 M, respectively) ( Figure 6A). 27 The same RI modification was introduced into thiorphan generating the end-group modified RI thiorphan,…”

“…Unlike the thiorphan, the end-group modified RI thiorphan cannot form this essential hydrogen bond with the acceptor in the active site of the ACE. 27 Like thiorphan, i.c.v. injection of end-group modified RI thiorphan resulted in potent antinociceptive activity in mice subjected to the hot plate and writhing tests.…”

“…These antinociceptive effects were blocked by pretreatment with naloxone, a potent opioid antagonist. 27 Interestingly, applying the RI modification to hydroxamates corresponding to the general formula HONHCO(CH 2 ) n CH(CH 2 Ph)CONHCH(CH 3 )CO 2 H, with n ϭ 0 or 1, which are potent inhibitors of three zinc metallopeptidases; neutral endopeptidase 24 -11, aminopeptidase N, and dipeptidylaminopeptidase generated some potent inhibitors of these enkephalindegrading enzymes. 29 These findings confirm the hypothesis that in some systems functional equivalence between peptides and their RI isomers reflect similar molecular topology at the active site and essentially equivalent hydrogenbonding interactions.…”

“…(A) Topological analogy between H-Phe-Ala-OH and its end-group modified RI analog H-gPhe-mAla-OH, both effective inhibitors of enkephalinase activity. 27 (B) Schematic illustration showing the relative disposition of peptide bonds in both orientations (normal a reversed) within the active site of thermolysin, a metalloendopeptidase with substrate specificity similar to that of enkephalinase. 28 Both thiorphan and its end-group modified RI analog display essentially equivalent hydrogen-bond interactions with Arg 203 and Asn 112 in the active site.…”

Effect of endopeptidase‐24.11 inhibitors and C‐ANP receptor ligand on responses evoked in arterioles of rat cremaster muscle by atrial natriuretic peptide

“…Thiorphan is a potent NEP inhibitor (Ki = 4 nM) but it is not completely specific because it also inhibits angiotensin-converting enzyme (ACE; Ki = 140 nM) (Roques et al, 1980). In contrast, its retro-inverso-isomer, retrothiorphan is almost as potent as thiorphan for NEP (Ki = 6 nM) with a very low affinity for ACE (IC50> 10 mM) (Roques et al, 1983). Finally kelatorphan is a potent inhibitor of both endopeptidase 24-11 (Ki = 1.8 nM) and aminopeptidase (Ki = 380 nM), and is unable to interact with ACE (IC5o > 10 mM) (Bouboutou et al, 1984).…”